Bottom Line:
The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen.Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Background: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth.

Methodology and principal findings: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.

Significance: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

pone-0009258-g005: TSLP is causative of both AD and MPD in N1N2K5 mice.TSLPR−/− mice were indistinguishable from wild type mice and therefore only the results of TSLPR−/− mice are shown. (A) Representative skin sections of TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for HE (upper panels), Ki67 (middle panels) and Toluidin blue (lower panels). Notch mutant mice lacking TSLPR have a markedly less proliferative epidermis, do not develop spongiosis and have large a reduction in dermal inflammatory cells (arrows, n = 6 per sample group, three individual experiments). (B) Myeloid cell counts in peripheral blood, and (C) spleen and lymph node (LN) macroscopy from TSLPR−/−, N1N2K5 and N1N2K5TSLPR−/− mice showing a rescue of the MPD phenotype. The bar diagrams represent mean values ± SD (n = 3 for each genotype of mice, three individual experiments). Representative flow cytometric analysis of myeloid and B cells of the spleen (D) and bone marrow (E) from TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for CD11b and Gr1, CD21 and CD23 (gated on B220+ splenic B cells), or B220 and CD43 (n = 6 per sample group, three individual experiments). [Scale bars: 50 µm].

Mentions:
We assessed whether excessive TSLP-mediated signaling is indeed responsible for the development of both the AD-like and MPD phenotypes. For this purpose TSLPR−/− mice [30] were intercrossed with N1N2K5 mice in order to generate triple mutants (N1N2K5 TSLPR−/−). As TSLPR−/− mice were identical to wild type mice in our analyses, we used the former as controls. Additional loss of TSLPR led to a markedly less proliferative epidermis, which was also devoid of spongiosis and displayed a substantial decrease in inflammatory dermal infiltrates compared to N1N2K5 animals (Figure 5A). Moreover, myeloid cell counts in peripheral blood from N1N2K5 TSLPR−/− mice were comparable to controls, while they were increased 5-fold in N1N2K5 mice (Figure 5B). N1N2K5 TSLPR−/− mice had normal sized spleens and lymph nodes (Figure 5C). FC analysis of splenocytes and BM cells of the three different genetic groups of mice showed that the absence of TSLPR in N1N2K5 mice was sufficient to restore normal hematopoiesis. The spleen and BM of N1N2K5 TSLPR−/− mice revealed the presence of normal granulocyte numbers and no block in B cell development (Figure 5D, E). These findings clearly reveal that both AD and the MPD are mediated by excessive TSLP levels.

pone-0009258-g005: TSLP is causative of both AD and MPD in N1N2K5 mice.TSLPR−/− mice were indistinguishable from wild type mice and therefore only the results of TSLPR−/− mice are shown. (A) Representative skin sections of TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for HE (upper panels), Ki67 (middle panels) and Toluidin blue (lower panels). Notch mutant mice lacking TSLPR have a markedly less proliferative epidermis, do not develop spongiosis and have large a reduction in dermal inflammatory cells (arrows, n = 6 per sample group, three individual experiments). (B) Myeloid cell counts in peripheral blood, and (C) spleen and lymph node (LN) macroscopy from TSLPR−/−, N1N2K5 and N1N2K5TSLPR−/− mice showing a rescue of the MPD phenotype. The bar diagrams represent mean values ± SD (n = 3 for each genotype of mice, three individual experiments). Representative flow cytometric analysis of myeloid and B cells of the spleen (D) and bone marrow (E) from TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for CD11b and Gr1, CD21 and CD23 (gated on B220+ splenic B cells), or B220 and CD43 (n = 6 per sample group, three individual experiments). [Scale bars: 50 µm].

Mentions:
We assessed whether excessive TSLP-mediated signaling is indeed responsible for the development of both the AD-like and MPD phenotypes. For this purpose TSLPR−/− mice [30] were intercrossed with N1N2K5 mice in order to generate triple mutants (N1N2K5 TSLPR−/−). As TSLPR−/− mice were identical to wild type mice in our analyses, we used the former as controls. Additional loss of TSLPR led to a markedly less proliferative epidermis, which was also devoid of spongiosis and displayed a substantial decrease in inflammatory dermal infiltrates compared to N1N2K5 animals (Figure 5A). Moreover, myeloid cell counts in peripheral blood from N1N2K5 TSLPR−/− mice were comparable to controls, while they were increased 5-fold in N1N2K5 mice (Figure 5B). N1N2K5 TSLPR−/− mice had normal sized spleens and lymph nodes (Figure 5C). FC analysis of splenocytes and BM cells of the three different genetic groups of mice showed that the absence of TSLPR in N1N2K5 mice was sufficient to restore normal hematopoiesis. The spleen and BM of N1N2K5 TSLPR−/− mice revealed the presence of normal granulocyte numbers and no block in B cell development (Figure 5D, E). These findings clearly reveal that both AD and the MPD are mediated by excessive TSLP levels.

Bottom Line:
The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen.Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Background: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth.

Methodology and principal findings: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.

Significance: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.